Autoimmune Disorders
In normal healthy individuals, B lymphocytes which comprise part of the body's humoral immune system help provide protection against infection by producing antibodies. Antibodies are proteins that attack invasive microbes or other “foreign” substances, removing these substances from the body and thus combating infection. Some foreign substances are proteins that provoke an immune response; i.e., the production of antibodies, and are termed antigenic proteins, also referred to herein as protein antigens. Antibodies produced by the immune system can be highly specific in binding and removing the particular antigens which provoke their production. In those individuals with a normally functioning immune system, a wide variety of antibodies are built up over time. These antibodies target and recognize various antigens which challenge the body's immune system, identifying particular antigens as either threatening or non-threatening to the organism.
Normally, the immune system readily distinguishes two general categories; recognizing antigenic proteins as either “self” or “not self.” “Self” antigens are typically recognized by the body's immune system as non-threatening, although this is not always the case. The body's immune system typically attacks only those antigens which are recognized as “not self.”
Autoimmune disorders are diseases that result from the body's production of an inappropriate immune response directed against its own “self” antigens. Such responses are frequently tissue directed or tissue specific. For example, the immune system may cease to recognize one or more of the body's normal proteins as “self”, creating antibodies termed autoantibodies, which target the body's own cells, tissues, and/or organs. The inflammation and damage caused by such attacks can lead to various autoimmune disorders.
The specific cause of autoimmune disease is unknown. Some evidence suggests that certain individuals have an inherited predisposition to develop autoimmune disease. Alternatively, some autoimmune disease, (e.g., rheumatic fever), appears to be triggered by bacterial or viral infection. The consequent immune response produces antibodies or T-cells which attack normal cells that display certain proteins that may structurally resemble (at least in part) the structure of certain proteins of the infecting microorganism(s).
Autoimmune disorders are categorized into two general types. Systemic autoimmune diseases are those diseases that cause damage to many organs. Localized, or tissue specific autoimmune diseases are those diseases which directly damage only a single organ or tissue. The distinctions between these two categories of disease, however, are not absolute. Frequently the deleterious effects of localized autoimmune disorders are not limited to the direct effects observed in a single organ or tissue, and may extend indirectly to other body organs and systems.
Common types of autoimmune disease include Rheumatoid arthritis (RA) and Juvenile RA (JRA) (joints; less commonly lung, skin), Type 1 Diabetes Mellitus (pancreas islets), Lupus [Systemic Lupus Erythematosus] (skin, joints, kidneys, heart, brain, red blood cells, other), Hashimoto's thyroiditis, Graves' disease (thyroid), Scleroderma (skin, intestine, less commonly lung), Celiac disease, Crohn's disease, Ulcerative colitis (GI tract), Sjogren's syndrome (salivary glands, tear glands, joints), Multiple sclerosis, Goodpasture's syndrome (lungs, kidneys), Addison's disease (adrenal), Wegener's granulomatosis (blood vessels, sinuses, lungs, kidneys), Primary biliary cirrhosis, Sclerosing cholangitis, Autoimmune hepatitis (liver), Polymyalgia Rheumatica (large muscle groups), Temporal Arteritis/Giant Cell Arteritis (arteries of the head and neck), and Guillain-Barre syndrome (nervous system).
Diagnosis of Autoimmune Disorder
The diagnosis of autoimmune disorders is generally carried out through a combination of clinical analysis of symptoms (presentation), blood tests to measure autoantibodies, inflammation and organ function, and other procedures including physical examination and/or X-rays.
Blood tests which measure autoantibodies are typically performed by detecting autoantibodies present in a serum sample taken from a subject having or suspected of having an autoimmune disorder. Commonly employed blood assays include Ouchterlony double immunodiffusion assays (DID) and counterimmunoelectrophoresis (CIE). These types of assays visualize precipitins (agglutinations) of autoantibody/antigen complexes formed by the binding of particular autoantibodies to their cognate antigens (Bunn & Kveder, 1993 Man. Biol. Mark. Dis. A3:1-12; Kluwer Academic). DID assays, for example, may be carried out in agar plates, with autoantigens placed in a central well symmetrically surrounded by peripheral wells. The peripheral wells are then loaded with test samples which contain antibodies that are diagnostic for a particular autoimmune disorder. The sample and test antigens are allowed to diffuse through the agarose, where agglutination of autoantibodies in the sample with a cognate autoantigen leads to the formation of an antibody/antigen complex. Extensive crosslinking of autoantibodies and autoantigens will occur in the agarose gel at a location where autoantibodies in the test sample and autoantigens from the peripheral wells reach equivalent concentrations due to diffusion. Such cross-linked complexes are termed precipitins. Precipitin formation is then visualized optically by direct light scattering of the autoantibody/test antigen complex.
Due to the specificity of binding of a given autoantibody for its cognate antigen, DID assays allow for positive detection of autoantibodies present in the sample being tested. Known DID assays, however, are non-quantitative and can suffer from a relative lack of sensitivity. In addition, relatively high numbers of autoantibodies in a sample are generally required for precipitin visualization. For these reasons, known DID assays may produce false negative results in some circumstances. Confirmation of a negative result returned from a DID assay, in view of clinical presentation of autoimmune disorder related symptoms requires additional tests be performed. This can result in additional expense, time lost before the initiation of any treatment regimen and general inconvenience to the subject.
Alternatively, autoantibodies present in a sample may be detected using a common biochemical technique called an enzyme-linked immunosorbent assay (ELISA), or enzyme immunoassay (EIA) (Charles P. J. et al. 1993 Man. Biol. Mark. Dis. A5:1-23; Kluwer Academic). In general terms, antibodies in a sample may be detected by ELISA as follows: An amount of a known antigen which binds specifically to a particular antibody is affixed to a surface, for example, the bottom of a microtitre plate well. A sample containing an unknown amount of the antibody to be detected is then placed into the well, allowing binding of the antigen to the antibody. The bound antibody is then detected using an anti-hIgG enzyme conjugate (anti-human immunoglobulin G).
Alternatively, a substrate for an enzyme bound to the antibody may be added to microtitre plate well, where activity of the enzyme converts this substrate to some type of detectable signal. The antibody may be covalently linked to an enzyme, or may be itself detected by use of an enzyme linked secondary antibody. Visualization of the detectable signal depends on the enzymatic substrate employed. Some ELISAs utilize chromogenic substrates, whereas other, more sensitive assays employ fluorogenic substrates. In the case of fluorescence ELISA, fluorescence detection of the antigen/antibody complexes allows the amount of antigen in the sample to be inferred by measuring the magnitude of the fluorescence.
An ELISA can also be used to detect autoantibodies in a blood sample obtained from a subject. Methods employing ELISA can detect very low concentrations of autoantibodies in a sample. However, ELISA methods of detection are prone to false positives due to the extreme sensitivity of the assay.
Consequently, there is a need for alternative methods of detecting autoantibodies in a sample suspected of containing autoantibodies that can provide a clinician with a more robust diagnostic tool available as a single test. A single test could provide the clinician greater confidence in his or her diagnosis without incurring the additional subject expense of further confirmatory testing, may be performed quickly and easily, and will also reduce the inconvenience to a subject of requiring further testing, potentially allowing an earlier initiation of the appropriate treatment regime.
It is an object of the present invention therefore, to at least go some way towards addressing the deficiencies of the prior art tests as outlined above by providing improved methods and/or compositions and/or kits that can be used to detect autoantibodies, and/or to at least provide the public with a useful choice.